The present invention relates to metabolic genes and their role in carcinogenesis. In particular, the invention relates to specified polymorphisms in genes encoding androgen-metabolic enzymes and their role in racial/ethnic susceptibility to prostate cancer.
Metastatic prostate cancer is a leading cause of cancer-related death in men. In the United States some 334,500 men are anticipated to be diagnosed during this year and over 41,800 to die from the disease (Parker, 1997). This cancer is characterized by a marked racial/ethnic variation in risk. African-American men have the highest prostate cancer incidence rate of any racial/ethnic group, which is two-thirds higher than for White males and more than twice as high as rates for Asian-Americans. However, despite its high prevalence, very little is known regarding genetic predisposition to prostate cancer. Recent biochemical, molecular and epidemiological evidence has produced widespread interest in the role of androgens in prostate cancer pathogenesis because of their important growth regulatory effects on prostate.
Steroid hormones are ubiquitous physiologic regulators that function by modulating gene expression. Their biosynthesis involves initial conversion of cholesterol to pregnenolone which then may be metabolized by a variety of pathways to yield progestins, mineralcorticoids, glucocorticoids, androgens, and estrogens. Androgens are required for normal sexual differentiation, growth and development, and the main sexual characteristics in men. The most abundant androgen, testosterone, is produced in Leydig cells involving cytochrome P450 enzymes. Testosterone can act directly on target cells, or it can be converted into its reduced more potent form, dihydrotestosterone, by the 5xcex1-reductase enzymes or to estradiol by the aromatase enzyme complex. Dihydrotestosterone forms a complex with the androgen receptor (AR), which translocates to the nucleus for transactivation of androgen-responsive genes and subsequent regulation of the growth of prostate cells. Dihydrotestosterone is inactivated by the 3-hydroxysteroid dehydrogenases, further modified and ultimately excreted (Coffey, 1993).
There are compelling reasons to believe that androgens play a central role in prostate carcinogenesis. The growth and maintenance of the prostate are dependent on androgens (Henderson, 1982). Prostate cancer regresses following ablative or antiandrogen therapy (Trunnel, 1950), and exogenous androgen supplementation is required in most animal prostate carcinogenesis models (Pollard, 1989; Shirai, 1995). Similarly, administration of steroid 5xcex1-reductase inhibitors, which diminishes DHT levels, results in a substantial decrease in prostatic secretion of the normal gland and a substantial increase in cell death in normal and transformed prostatic cells (Kadohama, 1984; Lamb, 1992). Racial populations with a higher incidence of prostate cancer were shown to have a higher activity of steroid 5xcex1-reductase (Lookingbill, 1991; Ross, 1992; Wu, 1995), and men with prostatic cancer have an increased conversion rate of testosterone to its reduced potent metabolite, dihydrotestosterone (Meikle, 1987).
Studies of the regulation of androgen biosynthesis in steroidogenic cells have focused on both transcriptional and post-translational regulation of the relevant proteins that catalyze these reactions such as the enzyme P450c17 (Picado-Leonard and Miller, 1987), the prostatic (or type II) steroid 5xcex1-reductase, and both the 3xcex2-hydroxysteroid dehydrogenase type II and the 17xcex2-hydroxysteroid dehydrogenase type III. Microsomal cytochrome P450c17 is encoded by the CYP17 locus and is the key branch point in human adrenal steroidogenesis. It mediates both 17 xcex1-hydroxylase and 17,20-lyase activities that are independently regulated (Miller, 1997). The former enzymatic activity leads to precursors of the glucocorticoid cortisol, whereas the latter activity yields precursors to the sex steroids (Brentano, 1990). Various mutations in the CYP17 gene are known that lead to deficiencies in either enzyme activity. Clinical phenotypes of these diseases include autosomal disorders producing an excess of mineralcorticoids and sexual differentiation abnormalities (Yamaguchi, 1997). Recent investigations identified a single base pair change in the 5xe2x80x2 region of the CYP17 gene creating an SP1-type (CCACC box) promoter site in which a thymidine (T) is replaced by a cytosine (C), 34 base pairs upstream from the initiation site of translation. The normal sequence has been designated as the A1 allele and the mutated sequence as the A2 allele (Carey, 1994). It was suggested that the additional promoter site influences promoter activity, thereby increasing levels of transcription leading to elevated synthesis of androgens (Carey, 1994).
Steroid 5xcex1-reductase acts on a variety of androgen responsive target tissues to mediate such diverse endocrine processes as male sexual differentiation in the fetus and prostatic growth in men. It also plays a role in several endocrine abnormalities. There are two isoforms of steroid 5xcex1-reductase, type I and type II, which are encoded by the SRD5A1 and SRD5A2 gene, respectively (Wilson, 1993; Labrie, 1992; Thigpen, 1992). Type I enzyme is expressed mostly in newborn scalp and in skin and liver and is primarily responsible for virilization and male pattern baldness. Type II enzyme is primarily expressed in genital skin and the prostate and is involved in prostate development and growth (Wilson, 1993). The entire cDNA sequence of human type II SRD5A2 has been determined (Andersson, 1991), and is reproduced here:
1 gcggccaccg gcgaggaaca cggcgcgatg caggttcagt gccagcagag cccagtgctg
61 gcaggcagcg ccactttggt cgcccttggg gcactggcct tgtacgtcgc gaagccctcc
121 ggctacggga agcacacgga gagcctgaag ccggcggcta cccgcctgcc agcccgcgcc
181 gcctggttcc tgcaggagct gccttccttc gcggtgcccg cggggatcct cgcccggcag
241 cccctctccc tcttcgggcc acctgggacg gtacttctgg gcctcttctg cgtacattac
301 ttccacagga catttgtgta ctcactgctc aatcgaggga ggccttatcc agctatactc
361 attctcagag gcactgcctt ctgcactgga aatggagtcc ttcaaggcta ctatctgatt
421 tactgtgctg aataccctga tgggtggtac acagacatac ggtttagctt gggtgtcttc
481 ttatttattt tgggaatggg aataaacatt catagtgact atatattgcg ccagctcagg
541 aagcctggag aaatcagcta caggattcca caaggtggct tgtttacgta tgtttctgga
601 gccaatttcc tcggtgagat cattgaatgg atcggctatg ccctggccac ttggtccctc
661 ccagcacttg catttgcatt tttctcactt tgtttccttg ggctgcgagc ttttcaccac
721 cataggttct acctcaagat gtttgaggac taccccaaat ctcggaaagc ccttattcca
781 ttcatctttt aaaggaacca aattaaaaag gagcagagct cccacaatgc tgatgaaaac
841 tgtcaagctg ctgaaactgt aattttcatg atataatagt catatatata tatatatata
901 tatatatata tatatatatg tatatatgta atagtaggtc tcctggcgtt ctgccagctg
961 gcctggggat tctgagtggt gtctgcttag agtttactcc tacccttcca gggaccccta
1021 tcctgatccc caactgaagc ttcaaaaagc cacttttcca aatggcgaca gttgcttctt
1081 agctattgct ctgagaaagt acaaacttct cctatgtctt tcaccgggca atccaagtac
1141 atgtggcttc atacccactc cctgtcaatg caggacaact ctgtaatcaa gaattttttg
1201 acttgaaggc agtacttata gaccttatta aaggtatgca ttttatacat gtaacagagt
1261 agcagaaatt taaactctga agccacaaag acccagagca aacccactcc caaatgaaaa
1321 ccccagtcat ggcttccttt ttcttggtta attaggaaag atgagaaatt attaggtaga
1381 ccttgaatac aggagccctc tcctcatagt gctgaaaaga tactgatgca ttgacctcat
1441 ttcaaatttg tgcagtgtct tagttgatga gtgcctctgt tttccagaag atttcacaat
1501 ccccggaaaa ctggtatggc tattcttgaa ggccaggttt taataaccac aaacaaaaag
1561 gcatgaacct gggtggctta tgagagagta gagaacaaca tgaccctgga tggctactaa
1621 gaggatagag aacagtttta caatagacat tgcaaactct catgtttttg gaaactggtg
1681 gcaatatcca aataatgagt agtgtaaaac aaagagaatt aatgatgagg ttacatgctg
1741 cttgcctcca ccagatgtcc acaacaatat gaagtacagc agaagcccca agcaactttc
1801 ctttcctgga gcttcttcct tgtagttctc aggacctgtt caagaaggtg tctcctaggg
1861 gcagcctgaa tgcctccctc aaaggacctg caggcagaga ctgaaaattg cagacagagg
1921 ggcacgtctg ggcagaaaac ctgttttgtt tggctcagac atatagtttt ttttttttta
1981 caaagtttca aaaacttaaa aatcaggaga ttccttcata aaactctagc attctagttt
2041 catttaaaaa gttggaggat ctgaacatac agagcccaca tttccacacc agaactggaa
2101 ctacgtagct agtaagcatt tgagtttgca aactcttgtg aaggggtcac cccagcatga
2161 gtgctgagat atggactctc taaggaaggg gccgaacgct tgtaattgga atacatggaa
2221 atatttgtct tctcaggcct atgtttgcgg aatgcattgt caatatttag caaactgttt
2281 tgacaaatga gcaccagtgg tactaagcac agaaactcac tatataagtc acataggaaa
2341 cttgaaaggt ctgaggatga tgtagattac tgaaaaatac aaattgcaat catataaata
2401 agtgtttttg ttgttcatta aataccttta aatcatg (SEQ ID NO: 1).
Germline mutations of the type II gene (SRD5A2) cause a rare human disorder, male psudohermaphroditism. Males with this disorder are phenotypically female at birth, but develop male musculature and other secondary sex characteristics at puberty (Wilson, 1993). The prostate, however, remains highly underdeveloped, and dihydrotestosterone levels are low despite a rise in testosterone in puberty, suggesting that these mutations are not implicated in prostatic diseases in adults (Thigpen, 1992). However, recent investigations have shown that the SRD5A2 gene may function as a candidate gene for predisposition to prostate cancer (Davis and Russell, 1993; Reichardt, 1995; Reichardt, 1996). Extensive genetic polymorphisms consisting of variable numbers of TA dinucleotide repeats have been identified in the 3xe2x80x2-untranslated region of the human SRD5A2 gene (Davis and Russell, 1993; Reichardt, 1995). Some of these polymorphisms have been shown to be unique to the highest prostate cancer risk population (African-Americans), suggesting the existence of a molecular genetic basis for the large difference in circulating levels of testosterone and the variation in racial/ethnic incidence of prostate cancer (Silver, 1994; Ross, 1995; Reichardt, 1995). Further examinations of the SRD5A2 gene revealed an additional polymorphism which was differentially distributed among racial/ethnic populations and appeared to determine in vivo steroid 5xcex1-reductase activity (Reichardt, 1996; Makridakis, 1997).
The 3-hydroxysteroid dehydrogenases are involved in the regulation of dihydroxytestosterone levels through inactivation of this metabolite (Coffey, 1993). Two isozyme forms of 3xcex2-hydroxysteroid dehydrogenase have been reported in humans. The type I enzyme is encoded by the HSD3B1 gene and is expressed mostly in the breast, placenta, and the skin (Labrie, 1992). The type II enzyme is encoded by the HSD3B2 gene and is primarily expressed in the adrenals, testis, and ovary (Labrie, 1992). Both genes have been cloned (Labrie, 1992) and a number of mutations in the HSD3B2 gene have been found to cause congenital adrenal hyperplasia, a rare human disorder (Rheaume, 1992). However, these and other mutations do not appear to be involved in prostatic cancer. In addition, a complex (TG)n, (TA)n (CA)n dinucleotide repeat polymorphism was identified in the third intron of the HSD3B2 gene, consisting of eight alleles (Verreault, 1994). Reactions of androgens and estrogens at the C-17 position, on the other hand, are catalyzed by 17xcex2-hydroxysteroid dehydrogenases. There have been four types (I-IV) reported and cloned which share less than 25% homology.
Current strategies to reduce the mortality rate of prostate cancer range from early detection using serologic testing for prostate-specific antigen (Catalona, 1991) to various therapeutic methods that interfere with androgen production and function (Aquilina, 1997). However, there are currently no genetic methods available for the diagnosis and prevention of prostate cancer. Thus, the identification of genetic polymorphisms controlling androgen biosynthesis and metabolism, that are responsible for predisposition of prostate cancer would provide for a better understanding of the mechanisms of cancer causation (including ethnic and individual susceptibility), and ultimately lead to ways of prostate cancer prevention. The present invention addresses these disadvantages present in the prior art.
The invention relates to polymorphisms in androgen-metabolic genes (SRD5A2, CYP17, HSD3B2 and HSD17B3) and to methods of using such mutations in the diagnosis and treatment of inheritable prostate cancer susceptibility.
One aspect of the invention relates to the identification of novel polymorphisms by hybridization, polymorphism and/or sequence analysis. Preferably, DNA is isolated from peripheral (blood) lymphocytes and analyzed for specific mutations by SSCP (single-strand conformation dependent DNA polymorphism) scanning and direct PCR (polymerase chain reaction) sequencing. The inheritance pattern of the specified gene polymorphisms are used to diagnose genetic susceptibility in men of various racial/ethnic populations who are genetically at increased risk for developing prostate cancer.
Other aspects of the invention include genetic probes comprising sequences complementary to the sequences containing the specified polymorphisms; cloning or expression vectors containing the nucleic acid sequences; host cells or organisms transformed with these expression vectors; methods for production and recovery of purified polypeptides from host cells; and the purified polypeptides themselves. Preferred embodiments include labeled binding agents, including antibodies, specific for the polypeptides encoded by the disclosed nucleic acids, which can be used to identify expression products of these diagnostic polymorphisms or alleles in patient derived fluid or tissue samples.
Yet other aspects of the invention relate to methods of using these nucleotide sequences or their complements, or fragments thereof, as hybridization probes, as oligomers for PCR, for chromosome and gene mapping, in the recombinant production of protein, and in generation of anti-sense DNA or RNA, their chemical analogs and the like.
For therapeutic intervention, the invention provides compositions which can functionally interfere with the transcription or translation products of the mutations and/or alleles within the specified androgen-metabolic genes associated with prostate cancer susceptibility. These include antisense nucleic acids, competitive peptides, encoded by the disclosed nucleic acids, and high affinity binding agents such as antibodies.